Events of the eukaryotic cell cycle are regulated by an evolutionarily conserved set of protein kinases. The cyclin-dependent kinases (Cdks) are important for driving cells through different phases of the cell cycle and their sequential activation and inactivation are tightly regulated. At the G2/M transition, activation of the mitotic Cdk, Cdc2, requires multiple events; these include the synthesis and binding of cyclin B, phosphorylation on Cdc2 at an activating site by Cak, and finally, Cdc25-dependent dephosphorylation of inactivating sites that have been phosphorylated by Wee1 and Myt1 (P. Nurse, Cell 79:547 (1994); R. W. King, P. K. Jackson, M. W. Kirschner, Cell 79:563 (1994); T. R. Coleman, W. G. Dunphy, Curr. Opin. Cell Biol. 6:877 (1994)).
How activation of a Cdk elicits the downstream events of cell cycle progression is less well understood. Activation of cyclin B/Cdc2 leads to the phosphorylation of a large number of proteins, mainly on sites containing a Ser/Thr-Pro motif. Protein phosphorylation is believed to alter the functions of proteins to trigger the events of mitosis. In a few cases, mitotic phosphorylation has been shown to regulate mitotic events (R. Heald and F. McKeon, Cell 61:579 (1990); E. Bailly, et al., Nature 350:715 (1991); A. Blangy, et al., Cell 83:1159 (1995)). However, it is not understood how the rapid changes in mitotic phosphorylation are converted to the sequential events of mitosis.
An important experimental tool which has uncovered the general role of phosphorylation in mitotic regulation is the MPM-2 monoclonal antibody (F. M. Davis, et al., Proc. Natl. Acad. Sci. USA 80:2926 (1983)). MPM-2 recognizes a Phospho.Ser/Thr-Pro epitope on approximately 50 proteins which are localized to various mitotic structures (J. M. Westendorf, P. N. Rao, L. Gerace, Proc. Natl. Acad. Sci. USA 91:714-8 (1994)). Several important mitotic regulators are recognized by this antibody, including Cdc25, Wee1, topoisomerase IIa, Cdc27, Map 4, INCENP and NIMA (Stukenberg, P. T., K. D. Lustig, T. J. McGarry, R. W. King, J. Kuang and M. W. Kirschner, Curr Biol 7:338-348 (1997)).
Currently six kinases have been shown to phosphorylate proteins in vitro to produce the MPM-2 epitope: Cdc2, Polo-like kinase (Plk1), NIMA, MAP kinase, a MAP kinase (MEK), and an unidentified activity ME-H (Kuang, J. and C. L. Ashorn., J Cell Biol 123:859-868 (1993); Taagepera et al., Mol Biol Cell 5:1243-1251 (1994); Kumagai, A. and W. G. Dunphy, Science 273:1377-1380 (1996); Renzi, L., M. S. Gersch, M. S. Campbell, L. Wu, S. A. Osmani and G. J. Gorbsky, J. Cell Sci 110:2013-2025 (1997)). However, these kinases also phosphorylate substrates that do not generate the MPM-2 epitope especially in cell cycle stages other than mitosis. This suggests that there are additional features that are required for the recognition by MPM-2. Determination of the optimal MPM-2 binding sequence have confirmed the importance of amino acid residues flanking the Phospho Ser/Thr-Pro motif for the MPM-2 recognition (Westendorf, J. M., P. N. Rao and L. Gerace. Proc Natl Acad Sci USA 91:714-718 (1994)). Westendorf, et al., 1994).
The present invention is based on the discovery that an essential mitotic peptidyl prolyl isomerase specifically recognizes phosphorylated serine/threonine-proline bonds present in mitotic phosphoproteins. As a result of this discovery, a novel class of moleculular compounds are available with activity to act as inhibitors of phosphoserine/phosphothreonine-proline specific peptidyl prolyl isomerases, in particular the peptidyl prolyl isomerase, Pin1, and other Pin1-like isomerases. Accordingly, these molecular inhibitors are useful to treat disorders of cell proliferation such as hyperplastic or neoplastic disorders, wherein treatment of the disorder with an inhibitor of the present invention results in the arrest of mitosis and apoptosis (cell death) of the target cells.
The inhibitor compounds of the present invention include any molecule that binds into the active site of the phosphoserine- or phosphothreonine-proline specific peptidyl prolyl isomerase and, upon binding to the isomerase, inhibits the isomerase activity. Encompassed by the present invention are inhibitor compounds that mimic the structure and conformation of the substrate moiety when bound to the catalytic site (also referred to herein as the active site) of the isomerase. Molecular inhibitors of the the present invention will typically have an inhibition constant (Ki) in the nanomolar to micromolar range. Specifically encompassed herein are organic molecules that mimic the structure and conformation of pSer/pThr and bind to the isomerase of interest, thereby inhibiting its activity.
The inhibitor compounds of the present invention inculde inhibitors that comprise a core region that mimics the pSer/pThr-Pro peptide moiety of the phosphoserine- or phosphothreonine-proline peptidyl prolyl isomerase substrate. Encompassed by the present invention are inhibitors that comprise the pSer/pThr mimic moiety with the mimic moiety being flanked on one side by hydrophobic groups and on the other side by hydrophobic or positively charged groups, wherein the groups would contact the active site of the isomerase of interest.
The inhibitor compounds of the present invention include compounds that contain a core sequence comprising xSer/xThrY wherein xe2x80x9cxxe2x80x9d is a negatively charged tetra- or pentavalent moiety and xe2x80x9cYxe2x80x9d is a Pro (proline) or a Pro analog. More specifically, the inhibitors of the present invention include compounds that inhibit a phosphoserine- or phosphothreonine-proline specific peptidyl-prolyl isomerase comprising a protein, polypeptide, peptide and/or a peptide mimetic wherein said protein, polypeptide, peptide or peptide mimetic comprises pSer/pThr. Specifically encompassed are inhibitors that have the core sequence of XXXpSer-pProXXX (SEQ ID NO.:1), wherein X is any L-amino acid or D-amino acid.
Candidate molecules of the present invention are evaluated for inhibitory activity in competitive inhibition assays. For example, the assay mixture would include the candidate molecule to be tested for inhibiting activity, the isomerase of interest and the intended substrate of the isomerase of interest. This admixture is maintained for a time sufficient and under conditions sufficient for the isomerase of interest to bind and catalyze the isomerization of its intended substrate. The catalytic activity of the isomerase of interest in the presence of the candidate inhibitor is then compared with the activity of the isomerase in the absence of the candidate inhibitor. If the activity of the isomerase in the presence of the inhibitor is less than the activity of the isomerase in the absence of the inhibitor, the candidate inhibitor is suitable for use as an inhibitor of the isomerase of interest.
Encompassed by the present invention are inhibitors of interphase-specific pSer/pThr-Pro specific peptidyl prolyl isomerases. Specifically encompassed by the present invention are inhibitors of the essential mitotic peptidyl prolyl isomerase, Pin1, and other PIN1-like isomerases.
Also encompassed by the present invention are methods of inhibiting mitotic peptidyl-prolyl isomerases comprising administering an effective amount of an inhibitor as described herein. For example, a composition comprising an effective amount of the inhibitor and a pharmaceutically acceptable carrier can be administered to an individual in need thereof. Specifically encompassed are methods of inhibiting unwanted cell growth resulting from a hyperplastic or neoplastic disorder. Also encompassed by the present invention are methods of inhibiting cell growth in target cells, comprising contacting the cells with an inhibitor as described herein.
The present invention also relates to libraries of peptides that comprises a mixture of substantially equimolar amounts of peptides comprising the sequence NH2-MAXXXpSXXXAKK (SEQ ID NO.:2), wherein for each peptide X is any amino acid.
The present invention also relates to methods of identifying a phosphorserine-or phosphothreonine-specific peptidyl prolyl isomerase inhibitor comprising the steps of:
a) providing a library of compounds that comprises a mixture of substantially equimolar amounts of peptides comprising the sequence X1X2X3pS-PX4X5X6, wherein for each peptide X is any amino acid;
b) contacting the library of a) with the peptidyl prolyl isomerase of interest under binding conditions for time sufficient for the isomerase to bind to the peptides;
c) determining the amino acid sequences of peptides bound to the isomerase of interest;
d) synthesizing the peptides of c); and
e) assaying peptides of d) for cis/trans isomerization by the peptidyl prolyl isomerase of interest to determine which peptides undergo isomerization by the isomerase of interest, thus identifying peptides that bind to the isomerase and are suitable for use as inhibitors of the isomerase of interest.
The present invention further relates to methods of identifying a phosphorserine or phosphothreonine-specific peptidyl prolyl isomerase inhibitor comprising the steps of:
a) providing the peptidyl prolyl isomerase of interest;
b) mixing the isomerase of interest with:
i) a candidate molecule and
ii) the substrate of the isomerase of interest to form an admixture of the isomerase of interest, candidate molecule and substrate;
c) maintaining the admixture of b) under conditions sufficient for the isomerase of interest to catalyze the cis/trans isomerization of the substrate; and
d) determining the Ki of the candidate molecule, wherein a Ki of 10 micromolar or less is indicative of an inhibitor of the peptidyl prolyl isomerase of interest.